Journal of the National Cancer Institute Advance Access originally published online on January 27, 2009
JNCI Journal of the National Cancer Institute 2009 101(3):194-204; doi:10.1093/jnci/djn477
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© The Author 2009. Published by Oxford University Press.
ARTICLES |
T670X KIT Mutations in Gastrointestinal Stromal Tumors: Making Sense of Missense
Affiliations of authors: Experimental Molecular Pathology, Department of Pathology (TN, EV, SP, ET), Department of Experimental Oncology (AG, MS), and Scientific Directorate (MAP), Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; Molecular Simulation Engineering Laboratory, DICAMP, University of Trieste, Trieste, Italy (GMP, MF, SP)
Correspondence to: Elena Tamborini, PhD, Unit of Experimental Molecular Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori Milano, Via G. Venezian 1, 20133 Milano, Italy (e-mail: elena.tamborini{at}istitutotumori.mi.it).
Background: Chronic myeloid leukemia, gastrointestinal stromal tumors (GISTs), and idiopathic hypereosinophilic syndrome are associated with pathological deregulation of the tyrosine kinases BCR-ABL, KIT, and PDGFRA, respectively. Patients who become resistant to imatinib treatment often develop secondary mutations, the most common of which results in a substitution of isoleucine for threonine at the same location in the ATP-binding domain in all three kinases (in KIT this occurs at amino acid 670). We sought to determine why Thr is always replaced by Ile.
Methods: All possible point mutations in the DNA triplet codon that could result in amino acid substitutions at Thr670 (Thr670Arg, Thr670Ile, Thr670Lys, Thr670Ala, Thr670Ser, Thr670Pro) were introduced by site-specific mutagenesis of the complementary DNA for a constitutively active, imatinib-sensitive form of the KIT receptor, 
559/KIT. The resulting mutant KIT proteins were transiently expressed in COS1 African green monkey kidney cells grown with and without imatinib, and cell extracts were analyzed for KIT activation by immunoprecipitation and immunoblotting to determine autophosphorylation levels. We also performed molecular modeling to estimate the relative affinities of wild-type (Thr670) KIT and the KIT mutants for ATP and imatinib.
Results: Like the parental strain, Thr670Ala, Thr670Ser, and Thr670Lys mutants were inhibited by 5 µM imatinib, but in comparison, they were only weakly active and Thr670Pro and Thr670Arg were not active at all. Only the Thr670Ile mutant was fully active (autophosphorylated) and resistant to imatinib. These findings were consistent with computer modeling predictions that ranked these mutants Thr 
Ile > Ala, Ser > Lys >> Pro according to their affinity for ATP but Thr > Ala, Ser > Lys >Pro Arg Ile according to their affinity for imatinib.
Conclusions: This combination of in vitro and molecular modeling analyses shows why, among all possible amino acid substitutions at position 670 of KIT, only Ile is naturally selected as a resistance mutant in imatinib-treated GIST patients.
| Context and Caveats Prior knowledge Patients with gastrointestinal stromal tumor, chronic myeloid leukemia, and idiopathic hypereosinophilic syndrome can develop resistance to imatinib by second-site mutation of the genes for the kinases KIT, BCR-ABL, and PDGFRA, respectively, which can cause an amino substitution homologous to the Thr670Ile mutant of KIT in all three kinases. Study design All possible point mutation-derived substitutions at the Thr670 codon were reconstructed using the complementary DNA for an activated version of KIT that also lacked amino acid 559. Mutant proteins were expressed in and immunoprecipitated from COS1 cells, and their activation/autophosphorylation in the presence and absence of imatinib was studied relative to the parental strain. All mutants were also examined by molecular modeling to calculate the relative free energies of ATP and imatinib binding. Contribution
The KIT mutants could be ranked according to their ATP-binding affinity as follows: Thr Implications This study provides a molecular explanation of why mutants like the Thr670Ile mutant of KIT are naturally selected in patients treated with imatinib. Limitations Not all mutants were expressed equally in COS cells, which could affect their relative autophosphorylation patterns. Only single nucleotide mutations in the KIT Thr670 codon were studied. From the Editors
|
Manuscript received March 25, 2008; revised November 13, 2008; accepted November 20, 2008.
Related Article in JNCI
CiteULike 
Connotea 
Del.icio.us What's this?
J Natl Cancer Inst 2009 101: 127.
This article has been cited by other articles:
|
|
 |
|
  E. Conca, T. Negri, A. Gronchi, E. Fumagalli, E. Tamborini, G. M. Pavan, M. Fermeglia, M. A. Pierotti, S. Pricl, and S. Pilotti Activate and resist: L576P-KIT in GIST Mol. Cancer Ther., September 1, 2009; 8(9): 2491 - 2495. [Abstract] [Full Text] [PDF]
|
|